All cellular processes are regulated by complex biochemical reactions. The first sign of a disease is often subtle biochemical changes in the reaction network. Multiplexed Foster resonant energy transfer (FRET) (H. J. Carlson and R. E. Campbell, “Genetically encoded FRET-based biosensors for multiparameter fluorescence imaging,” Current Opinion in Biotechnology 20, 19 (2009)) imaging provides a systematic way to study complex biochemical processes. At present, real-time studying of complex cellular processes is limited by our inability to study the FRET network among multiple fluorescent labels simultaneously in live cells and animals. The difficulty rises from the complex photon pathway network in a multi-label FRET complex. To apply multiplexed FRET image in live imaging, all photon pathways need to be imaged in parallel (M. Zhao, R. Huang, and L. Peng, “Quantitative multi-color FRET measurements by Fourier lifetime excitation-emission matrix spectroscopy,” Optics Express (2012)).
First established by Theodor Förster in the 1940s (T. Forster, “Zwischenmolekulare energiewanderung and fluoreszenz,” Annalen Der Physik 2, 55-75 (1948)), Förster resonant energy transfer (FRET) is widely used as a fluorescence spectroscopy method to measure distances between fluorophores on the nanometer scale. FRET occurs when an excited donor fluorophore transfers its energy to an adjacent ground-state acceptor fluorophore through dipole coupling. Through the FRET process, the donor emission is quenched and the acceptor emission is enhanced. This process depends strongly on the distance between molecules in the 1-10 nm range, and can therefore be exploited as a “spectroscopic ruler” (L. Stryer, “Fluorescence energy-transfer as a spectroscopic ruler,” Annual Review of Biochemistry 47, 819-846 (1978)). With recent advances in fluorescence proteins, organic dyes and instrumentation, FRET has found an ever increasing range of applications in biological studies, ranging from tracking protein-protein interactions in cellular processes (M. Elangovan, R. N. Day, and A. Periasamy, “Nanosecond fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy to localize the protein interactions in a single living cell,” Journal of Microscopy-Oxford 205, 3-14 (2002)), probing DNA/RNA regulations and dynamics (S. C. Blanchard, H. D. Kim, R. L. Gonzalez, J. D. Puglisi, and S. Chu, “tRNA dynamics on the ribosome during translation,” Proceedings of the National Academy of Sciences of the United States of America 101, 12893-12898 (2004)), to high-throughput drug screening (S. Kumar, D. Alibhai, A. Margineanu, R. Laine, G. Kennedy, J. McGinty, S. Warren, D. Kelly, Y. Alexandrov, I. Munro, C. Talbot, D. W. Stuckey, C. Kimberly, B. Viellerobe, F. Lacombe, E. W. F. Lam, H. Taylor, M. J. Dallman, G. Stamp, E. J. Murray, F. Stuhmeier, A. Sardini, M. Katan, D. S. Elson, M. A. A. Neil, C. Dunsby, and P. M. W. French, “FLIM FRET technology for drug discovery: automated multiwell-plate high-content analysis, multiplexed readouts and application in situ,” Chemphyschem 12, 609-626 (2011)). Most FRET biological studies were carried out with two different fluorophores, of which the donor and acceptor can either be fused with a flexible linker (single-chain FRET) or fused to two different molecules respectively (dual chain FRET). Single-chain FRET is used to detect conformation change in the flexible linker. Dual-chain FRET is used to detect interactions between two molecules.
Fluorescent signals from a FRET system can be represented in terms of excitation-emission matrix (EEM) channels, which are characterized by their individual exciters (which fluorophore absorbs the excitation photon) to emitters (which fluorophore emits the fluorescence photon) pathways. For two-color FRET, three possible EEM channels exist: excitation of the donor and its subsequent fluorescence emission (donor EEM channel or donor self excitation-emissions pathway); excitation of the acceptor and its subsequent emission (acceptor EEM channel or acceptor self excitation-emissions pathway); and excitation of the donor, which excites the acceptor via FRET, followed by emission from the acceptor (FRET EEM channel or FRET pathway). For FRET involving more than two colors, more EEM channels exist, as shown in FIG. 1.
FIGS. 1(a) and 1(b) illustrate an excitation emission matrix (EEM) representation of three-color FRET between fluorescein, Cy3 and Cy5. FIG. 1(a) shows photon pathways in a three-color FRET process. Six possible exciter-to-emitter photon pathways are present: three self excitation-emission EEM channels with Fluorescein, Cy3 and Cy5 (e11, e22 and e33 illustrated in FIG. 1(b)), and three FRET EEM channels (e12, e13 and e23 illustrated in FIG. 1(b)). Each of the three non-FRET EEM channels (e11, e22 and e33 illustrated in FIG. 1(b)) defines a self excitation-emission decay photon pathway. The FRET EEM channel e12 is a photon pathway in which photons emitted by donor fluorophores in response to the 488 nm radiation and transfer energy to acceptor fluorophores, which then emit photons of wavelengths in the 550 to 600 nm range as shown in FIG. 1(b), thus defining a FRET photon pathway. This pathway is marked e12 in FIG. 1(b). The same is true for FRET EEM channels e13 and e23. Thus each of the three FRET EEM channels (e12, e13 and e23 illustrated in FIGS. 1(a) and 1(b)) defines a donor excitation-acceptor emission decay photon pathway. FIG. 1(b) is an EEM representation of the three-color FRET as a function of both excitation and emission wavelengths. Different photon pathways occupy different regions of the EEM. For each photon pathway, the excitation spectrum follows the exciter, and the emission spectrum follows the emitter.
Note that in FIG. 1(b), each of the graphical plots depict emissions of the same intensity. Thus, as shown in FIG. 1(b), there are overlaps between the donor EEM channels of Fluorescein and Cy3 (e11 and e22 in FIG. 1(b)) and a FRET EEM channel from donor Fluorescein to an Acceptor (e12 in FIG. 1(b)). There is therefore bleed-through between the two donor channels and the FRET EEM channel. While not shown in FIG. 1(b), there may be additional bleed-through between the channels.
To quantify the absolute FRET efficiency, which is the probability of energy transfer from a donor to an acceptor, the current standard practice is to apply fluorescence lifetime imaging (FLIM) (H. Wallrabe and A. Periasamy, “Imaging protein molecules using FRET and FLIM microscopy,” Current Opinion in Biotechnology 16, 19-27 (2005)), a time-resolved fluorescence method, on the donor EEM channel, where the quenching effect of a FRET process causes a lifetime decrease according to equation (1) below.τDDonorEEM=(1−η)τ0  (1)where τ0 is the donor lifetime without FRET, and η is the FRET efficiency.
In other words, the donor fluorophores involved in a FRET process transfer energy to acceptor fluorophores, causing the donor fluorophores to lose energy in this FRET process and to return to the ground state faster than through the donor EEM channel alone.
Such practice is unsuitable for analyzing multi-color FRET, where multiple FRET processes can affect the lifetime of a donor in the same time, and the donor lifetime alone cannot distinguish different processes. Furthermore, current FLIM techniques still have inferior imaging performances in 3D spatial resolution, speed, and multiplexing ability. 3D point scanning FLIM with either time domain (W. Becker, A. Bergmann, M. A. Hink, K. Konig, K. Benndorf, and C. Biskup, “Fluorescence lifetime imaging by time-correlated single-photon counting,” Microscopy Research and Technique 63, 58-66 (2004)) or frequency domain (P. T. C. So, T. French, W. M. Yu, K. M. Berland, C. Y. Dong, and E. Gratton, “Time-resolved fluorescence microscopy using two-photon excitation,” Bioimaging 3, 49-63 (1995)) methods are too slow for live imaging. Wide-field FLIM has a faster frame rate, but does not have native 3D section ability, and requires special optical sectioning techniques such as structured illumination (M. J. Cole, J. Siegel, S. E. D. Webb, R. Jones, K. Dowling, P. M. W. French, M. J. Lever, L. O. D. Sucharov, M. A. A. Neil, R. Juskaitis, and T. Wilson, “Whole-field optically sectioned fluorescence lifetime imaging,” Optics Letters 25, 1361-1363 (2000)) or spinning disc confocal microscopy (D. M. Grant, J. McGinty, E. J. McGhee, T. D. Bunney, D. M. Owen, C. B. Talbot, W. Zhang, S. Kumar, I. Munro, P. M. P. Lanigan, G. T. Kennedy, C. Dunsby, A. I. Magee, P. Courtney, M. Katan, M. A. A. Neil, and P. M. W. French, “High speed optically sectioned fluorescence lifetime imaging permits study of live cell signaling events,” Optics Express 15, 15656-15673 (2007)) for 3D imaging, which significantly increases acquisition time and instrumentation complexity. More importantly, existing FLIM techniques are not multiplexing friendly, especially in multi-laser excitation imaging. While emission-multiplexed FLIM can be implemented through multiple detectors (W. Becker, A. Bergmann, and C. Biskup, “Multispectral fluorescence lifetime imaging by TCSPC,” Microscopy Research and Technique 70, 403-409 (2007)) or hyperspectral imaging (P. De Beule, D. M. Owen, H. B. Manning, C. B. Talbot, J. Requejo-Isidro, C. Dunsby, J. McGinty, R. K. P. Benninger, D. S. Elson, I. Munro, M. J. Lever, P. Anand, M. A. A. Neil, and P. M. W. French, “Rapid hyperspectral fluorescence lifetime imaging,” Microscopy Research and Technique 70, 481-484 (2007)), previous excitation-multiplexed FLIM techniques use a time-sharing scheme on multiple excitation wavelengths (T. A. Laurence, X. X. Kong, M. Jager, and S. Weiss, “Probing structural heterogeneities and fluctuations of nucleic acids and denatured proteins,” Proceedings of the National Academy of Sciences of the United States of America 102, 17348-17353 (2005); D. M. Owen, E. Auksorius, H. B. Manning, C. B. Talbot, P. A. A. de Beule, C. Dunsby, M. A. A. Neil, and P. M. W. French, “Excitation-resolved hyperspectral fluorescence lifetime imaging using a UV-extended supercontinuum source,” Optics Letters 32, 3408-3410 (2007)), which requires specialized laser sources with sophisticated laser control and further prolongs the already slow FLIM image acquisition.
It is therefore desirable to provide improved systems able to measure time-resolved excitation-emission fluorescence signals from a sample, where the above short comings are alleviated.